Direct, in situ selective detection of intracellular formaldehyde (FA) is of great significance for understanding its function in FA-related diseases. Herein, red carbon dots (RCD) are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA. Upon addition of FA, the -NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water. In addition, the nanoprobes exhibit outstanding photostability, rapid response (<1 min), high sensitivity (～9.9μmol/L) and excellent selectivity toward FA over other aldehyde group compounds. Notably, owing to the good cell-membrane permeability and biocompati-bility, as well as the large two-photon absorption cross-section, the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation. Moreover, RCD could stain the tissue of zebrafishes at depths interval of up to 240μm under two-photon excitation. This research implied that RCD are promising tools for directly and in situ imaging FA in vivo, thus providing critical insights into FA-related pathophysiological processes.